The goal of this project is to facilitate research of the molecular structure of HIV-1 integrase (IN), a virallyencoded enzyme that is essential for HIV-1 replication. Integrase is a promising molecular target for rational drug design, but there is currently no structural data on full length or substrate-bound IN. The integrase is known to be a conformationally dynamic protein, and current evidence indicates that it is capable of adopting the defined conformation only upon binding to its DMA substrate(s) and metal cofactors. We propose to obtain stabilized IN-DNA complexes suitable for structural and biochemical studies of HIV-1 and ASV integrase by chemically crosslinking the protein to nucleic acid, without distorting their structures. First, it is necessary to identify the sites of preferred IN-DNA interactions. This will be achieved by the method of photoaffinity crosslinking with diazirine-based heterobifunctional reagents. The information about the sites of contacts with several DNA substrates representing separate stages in IN enzymatic activities will be used to synthesize modified DNAs with sulfhydryl groups placed at specific locations to form S-S bonds with the corresponding novel Cys IN derivatives. IN-DNA complexes will be prepared in quantities sufficient for structural and biochemical studies in the laboratories of our collaborators (Dr. Alexander Wlodawer in NCI-Frederick and Dr. Anna-Marie Skalka in Fox Chase Cancer Center, Philadelphia). Comparison of the structure and properties of HIV-1 IN and those of the homologous enzyme for the avian sarcoma virus (ASV) is a key element of the experimental strategy of this work. As these enzymes catalyze identical reactions, but at different rates and recognizing slightly different substrates, such analyses can reveal common features intrinsic to the chemistries utilized by both enzymes, as well as features that determine viral specificity. Structural information gained in these studies should bring invaluable insight into mechanisms of functioning of the retroviral integrase and facilitate the development of new antiretrovirals.